LOCUS       sequence-351711-        8347 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of PKC-delta-eDHFR(69K6) chimera fused to 
            miRFP670.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8347)
  AUTHORS   Suzuki S, Nakamura A, Hatano Y, Yoshikawa M, Yoshii T, Sawada S, 
            Atsuta-Tsunoda K, Aoki K, Tsukiji S
  TITLE     A chemogenetic platform for controlling plasma membrane signaling 
            and synthetic signal oscillation.
  JOURNAL   Cell Chem Biol. 2022 Sep 15;29(9):1446-1464.e10. doi: 
            10.1016/j.chembiol.2022.06.005. Epub 2022 Jul 13.
  PUBMED    35835118
REFERENCE   2  (bases 1 to 8347)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.chembiol.2022.06.005"; journalName: "Cell Chem Biol"; 
            date: "2022-09-15- 15"; volume: "29"; issue: "9"; pages: 
            "1446-1464.e10"
COMMENT     Sequence Label: pCAGGS-chPKCdelta/eDHFR(69K6)-miRFP670
FEATURES             Location/Qualifiers
     source          1..8347
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          127..1143
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1067..1089
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1151..1170
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1221..1230
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3744..4691
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     primer_bind     complement(4710..4730)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4710..4728)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4749..4765)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4867..4886)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4932..4987
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4986..5005)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5348..5364)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5372..5388
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5396..5402,5403..5420,5421..5426))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5441..5462
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5521..5716
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5567..5702
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5722..5856
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5759..5778)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5813..5832
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5924..5941)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6095..6683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6175..6194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6854..7645,7646..7714))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7477..7496
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7715..7819)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        7850..8229
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
       61 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
      121 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
      181 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
      241 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
      301 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
      361 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
      421 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
      481 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
      541 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
      601 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
      661 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
      721 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
      781 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
      841 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
      901 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
      961 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     1021 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     1081 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     1141 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattcata
     1201 gtagctagca gttgctcgag gccaccatgg cgccgttcct gcgcatcgcc ttcaactcct
     1261 atgagctggg ctccctgcag gccgaggacg aggcgaacca gcccttctgt gccgtgaaga
     1321 tgaaggaggc gctcagcaca gagcgtggga aaacactggt gcagaagaag ccgaccatgt
     1381 atcctgagtg gaagtcgacg ttcgatgccc acatctatga ggggcgcgtc atccagattg
     1441 tgctaatgcg ggcagcagag gagccagtgt ctgaggtgac cgtgggtgtg tcggtgctgg
     1501 ccgagcgctg caagaagaac aatggcaagg ctgagttctg gctggacctg cagcctcagg
     1561 ccaaggtgtt gatgtctgtt cagtatttcc tggaggacgt ggattgcaaa cagtctatgc
     1621 gcagtgagga cgaggccaag ttcccaacga tgaaccgccg cggagccatc aaacaggcca
     1681 aaatccacta catcaagaac catgagttta tcgccacctt ctttgggcaa cccaccttct
     1741 gttctgtgtg caaagacttt gtctggggcc tcaacaagca aggctacaaa tgcaggcaat
     1801 gtaacgctgc catccacaag aaatgcatcg acaagatcat cggcagatgc actggcaccg
     1861 cggccaacag ccgggacact atattccaga aagaacgctt caacatcgac atgccgaccg
     1921 gttctgggtc cgcagccagt agcgccggaa gtatgatcag tctgattgcg gcgttagcgg
     1981 tagatcgcgt tatcggcatg gaaaacgcca tgccgtggaa cctgcctgcc gatctcgcct
     2041 ggtttaaacg caacacctta aataaacccg tgattatggg ccgccatacc tgggaatcaa
     2101 tcggtcgtcc gttgccagga cgcaaaaata ttatcctcag cagtcaaccg ggtacggaca
     2161 aaaaaaagaa aaagaaagat cgcgtaacgt gggtgaagtc ggtggatgaa gccatcgcgg
     2221 cgtgtggtga cgtaccagaa atcatggtga ttggcggcgg tcgcgtttat gaacagttct
     2281 tgccaaaagc gcaaaaactg tatctgacgc atatcgacgc agaagtggaa ggcgacaccc
     2341 atttcccgga ttacgagccg gatgactggg aatcggtatt cagcgaattc cacgatgctg
     2401 atgcgcagaa ctctcacagc tattgctttg agattctgga gcggcggggt gcttcgggtg
     2461 gcggcggatc ttcagctccc gggggcatca accagaagct tttggctgag gccttgaacc
     2521 aagtcaccca gagagcctcc cggagatcag actcagcctc ctcagagcct gttgggatat
     2581 atcagggttt cgagaagaag accggagttg ctggggagga catgcaagac aacagtggga
     2641 cctacggcaa gatctgggag ggcagcagca agtgcaacat caacaacttc atcttccaca
     2701 aggtcctggg caaaggcagc ttcgggaagg tgctgcttgg agagctgaag ggcagaggag
     2761 agtactttgc catcaaggcc ctcaagaagg atgtggtcct gatcgacgac gacgtggagt
     2821 gcaccatggt tgagaagcgg gtgctgacac ttgccgcaga gaatcccttt ctcacccacc
     2881 tcatctgcac cttccagacc aaggaccacc tgttctttgt gatggagttc ctcaacgggg
     2941 gggacctgat gtaccacatc caggacaaag gccgctttga actctaccgt gccacgtttt
     3001 atgccgctga gataatgtgt ggactgcagt ttctacacag caagggcatc atttacaggg
     3061 acctcaaact ggacaatgtg ctgttggacc gggatggcca catcaagatt gccgactttg
     3121 ggatgtgcaa agagaacata ttcggggaga gccgggccag caccttctgc ggcacccctg
     3181 actatatcgc ccctgagatc ctacagggcc tgaagtacac attctctgtg gactggtggt
     3241 ctttcggggt ccttctgtac gagatgctca ttggccagtc ccccttccat ggtgatgatg
     3301 aggatgaact cttcgagtcc atccgtgtgg acacgccaca ttatccccgc tggatcacca
     3361 aggagtccaa ggacatcctg gagaagctct ttgaaaggga accaaccaag aggctgggag
     3421 tgacgggaaa catcaaaatc caccccttct tcaagaccat aaactggact ctgctggaaa
     3481 agcggaggtt ggagccaccc ttcaggccca aagtgaagtc acccagagac tacagtaact
     3541 ttgaccagga gttcctgaac gagaaggcgc gcctctccta cagcgacaag aacctcatcg
     3601 actccatgga ccagtctgca ttcgctggct tctcctttgt gaaccccaaa ttcgagcacc
     3661 tcctggaaga tgcggccgct ggtagtggtg ctggctctgg tgctggtagt ggcgctggtt
     3721 ccggtgctgg ctcttcggcc gctatggtag caggtcatgc ctctggcagc cccgcattcg
     3781 ggaccgcctc tcattcgaat tgcgaacatg aagagatcca cctcgccggc tcgatccagc
     3841 cgcatggcgc gcttctggtc gtcagcgaac atgatcatcg cgtcatccag gccagcgcca
     3901 acgccgcgga atttctgaat ctcggaagcg tactcggcgt tccgctcgcc gagatcgacg
     3961 gcgatctgtt gatcaagatc ctgccgcatc tcgatcccac cgccgaaggc atgccggtcg
     4021 cggtgcgctg ccggatcggc aatccctcta cggagtactg cggtctgatg catcggcctc
     4081 cggaaggcgg gctgatcatc gaactcgaac gtgccggccc gtcgatcgat ctgtcaggca
     4141 cgctggcgcc ggcgctggag cggatccgca cggcgggttc actgcgcgcg ctgtgcgatg
     4201 acaccgtgct gctgtttcag cagtgcaccg gctacgaccg ggtgatggtg tatcgtttcg
     4261 atgagcaagg ccacggcctg gtattctccg agtgccatgt gcctgggctc gaatcctatt
     4321 tcggcaaccg ctatccgtcg tcgactgtcc cgcagatggc gcggcagctg tacgtgcggc
     4381 agcgcgtccg cgtgctggtc gacgtcacct atcagccggt gccgctggag ccgcggctgt
     4441 cgccgctgac cgggcgcgat ctcgacatgt cgggctgctt cctgcgctcg atgtcgccgt
     4501 gccatctgca gttcctgaag gacatgggcg tgcgcgccac cctggcggtg tcgctggtgg
     4561 tcggcggcaa gctgtggggc ctggttgtct gtcaccatta tctgccgcgc ttcatccgtt
     4621 tcgagctgcg ggcgatctgc aaacggctcg ccgaaaggat cgcgacgcgg atcaccgcgc
     4681 ttgagagcta ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     4741 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctaattca ctcctcaggt
     4801 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca
     4861 ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct
     4921 gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg
     4981 tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt
     5041 ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata
     5101 aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc
     5161 cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc
     5221 cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc
     5281 agtcatagct gtccctcttc tcttatggag atccctcgac ctgcagccca agcttggcgt
     5341 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5401 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5461 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc
     5521 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     5581 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     5641 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     5701 cctaggcttt tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc
     5761 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5821 tccaaactca tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac
     5881 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5941 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6001 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6061 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6121 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6181 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6241 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6301 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6361 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6421 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6481 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6541 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6601 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6661 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6721 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6781 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6841 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6901 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6961 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7021 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7081 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7141 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7201 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7261 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7321 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7381 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7441 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7501 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7561 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7621 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7681 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7741 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7801 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgggtcg acattgatta
     7861 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     7921 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     7981 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     8041 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     8101 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     8161 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     8221 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
     8281 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     8341 ggggggg
//
